Cerebrum

Editor’s Note: The circadian rhythm—the 24-hour cycle of the physiological processes of living beings—is instrumental in determining the sleeping and feeding patterns of all animals, including humans. Clear patterns of brain-wave activity, hormone production, cell regeneration, and other biological activities are linked to this daily cycle. Our author focuses on two relatively new areas of research—circadian genomics and epigenomics—and their potential for advancing medical insight.

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Each morning we wake up from a night of sleep, and each day we eat
our regularly timed meals, go through our normal routines, and fall asleep
again for another night. This rhythm, so-called circadian—after the Latin words
circa diem (“about a day”)—underlies a wide variety
of human physiological functions, including sleep-wake cycles, body
temperature, hormone secretion, locomotor activity, and feeding behavior.

A simple look at other organisms reveals that
circadian rhythms are remarkably conserved throughout evolution. Whether we
consider the cyclic movements of leaves on a plant, the activities of a house
cat, or the morning singing of birds, all follow a daily cycle that, being so
natural and ancient, generally happens at a subconscious level. Over the past
several decades, researchers have described a plethora of cyclic behaviors,
metabolic rhythms, and physiological oscillations—all following a circadian
pattern. Scientists have observed these behaviors in organisms as different as
fungi, insects, unicellular protists, plants, cyanobacteria, vertebrates, and
mammals. The rhythms are so wide-ranging that they include both the gravity-driven
orientation of the photosynthetic flagellate Euglena gracilis and the social behavior of mammals in a group.

Why are circadian rhythms so omnipresent? The
answer is straightforward. These biological cycles are based on the most
ancient feature of our environment: the astronomical rotation of Earth on its
axis, leading to the daylight-darkness cycle—the rhythmic repetition of days
and nights.1,2This feature has remained immutable over a
billion years—although the length of the photoperiod has shortened somewhat
over time.1

Scientists generally think that living beings have
developed by adapting to the daylight-darkness cycle. My personal view is that,
in addition to the adaptation process, life has developed because of the 24-hour light-dark cycle. Life-forms and their
cellular, organismal, and molecular features would have been completely
different on a planet with a longer or shorter light-dark cycle. Simple
experiments on the small flowering plant Arabidopsis
thaliana show that its size is reduced by half when subjected to light-dark
cycles of 20 hours or 28 hours, corresponding to a planetary rotation that is
only one-fifth slower or faster than Earth’s.3

The role of the circadian clock appears to be so
fundamental that, as shown in a number of studies, it has intimate links with
the cell cycle.4 This is nicely illustrated when we consider
evolution’s role in the process. Indeed, the cell division of a number ofunicellular organisms, such as the greenalga Chlamydomonas reinhardtii,
the cyanobacteriumSynechococcus
elongates, and the dinoflagellate Gonyaulax
polyedra, can be timed by a circadian mechanism. Also, disruption of the
clock may have drastic health consequences. In humans, for example, night-shift
workers have increased incidence of metabolic disorders.

In the past two decades the knowledge in the
field of circadian biology has increased remarkably, such that today it is safe
to claim that circadian rhythms represent possibly the ultimate example of
systems biology. Some of these fairly recent findings, in my view, have
prominently shaped our modern view of the field.

My First Encounter

I attended my very first conference on circadian
rhythms more than 20 years ago. I was invited because, while working on the
relationship between a messenger important in many biological processes and a
gene (cyclic-AMP responsive element modulator, or CREM), my team stumbled on a
clever molecular mechanism that allows expression to be cyclic in the pineal
gland. Subsequently, we determined that CREM transcriptionally controls the
gene encoding the serotonin N-acetyltransferase, an enzyme responsible for the
rhythmic synthesis of the hormone melatonin from the pineal gland.

For the most part, the conference was a series
of descriptive presentations about measuring circadian oscillations in a wide
variety of organisms and physiological settings. Coming from the hard-core field
of molecular transcription, I was fascinated by the spectacular variety of
biological systems presented and intrigued by the obvious opportunities for
mechanistic investigation. Most important, I found (and still find) the
self-sustaining nature of circadian rhythmicity thought provoking. The field
was on the verge of witnessing a series of conceptual transformations.

What is the
evolutionary advantage of circadian clocks? They allow organisms to anticipate
daily events (for example, food availability and predator pressure for animals,
and sunrise for plants) rather than just reacting to them. Because the measure
of time by circadian pacemakers is only approximate, their phase needs to be
adjusted daily to stay in synchrony with geophysical time. Self-evident even to
nonspecialists, light is the dominant entraining cue for all circadian
timekeepers and is consequently considered the most critical zeitgeber (German for “time giver”) for circadian physiology. In
mammals, the anatomical structure that governs circadian rhythms is the
suprachiasmatic nucleus (SCN), a small area in the brain consisting of approximately
15,000 neurons localized in the anterior hypothalamus.

For decades scientists have considered this
central pacemaker to be the unique circadian clock controlling all daily
behavior, metabolism, and physiology.1, 5 SCN neurons are able to
self-sustain rhythmicity for weeks even when isolated in a culture dish. Their
plasticity is also remarkable: The SCN is reset every day by the light-dark
cycle, and thereby undergoes seasonal variations corresponding to the changes
in the photoperiod. Thus, SCN neurons are wired to oscillate (to repeatedly
move in one direction and back many times), but they receive the light signal
through specialized retinal neurons and via the retinal-hypothalamic tract
(RHT), thereby insuring their timely adjustment to the changing photoperiod and
environment.5 The SCN’s role as the master clock is demonstrated by
grafting experiments: Normal SCN grafted into a genetically arrhythmic animal
can restore circadian rhythmicity.

Clocks Everywhere!

One
discovery that has deeply affected the field of circadian biology during the
past 15 years is that oscillators are present in most tissues. The thinking for
decades was that the SCN alone directs all circadian body functions, but more
recent findings reveal that the liver, spleen, muscle, and other body functions
all have their own internal clock. My research team first described this
finding in a vertebrate,6 extending previous observations made in Drosophila.7, 8 Soon afterward,
this finding was confirmed in mammals.2, 9In Drosophila and zebra fish, light-dark
cycles can directly entrain all oscillators, a scenario that is possible only
in organisms in which at least some photons reach the internal organs.6, 7

The
evolution of larger and thus opaque organisms necessitated the development of a
different, nonphotic (in addition to photic) communication system. In mammals, we
see this in the organization of neuroendocrine circuits that convey the timing
information from the SCN to the entire organism via direct and indirect
signaling pathways. The SCN thereby functions as a master pacemaker, a kind of
orchestra director that hierarchically coordinates
the subsidiary oscillators located in peripheral tissues.

This
notion was further illustrated by an experiment in which fibroblasts (a type of
cell that plays a critical role in wound healing) that originated from a mutant
mouse (and thereby had a faster clock) took on the rhythm of a host mouse when
grafted as a subcutaneous implant.10Additional evidence demonstrated the presence of circadian
oscillators even in established cell lines: In cultured fibroblasts the
endogenous clock system needed a simple serum shock to be resynchronized,11
while the pacemaker of zebra fish’s embryonic cells started ticking upon
exposure to a short pulse of light.2Together, these findings significantly extended
our view of circadianorganization
at the whole-organism level. They also underscored the fact that circadian-clock
functions are not the prerequisite of a relatively small number of SCN neurons,
as scientists thought for decades, but instead are common features of most
cells.

Yet, more than a decade after these
discoveries, some fundamental questions remain unanswered. Specifically, how do SCN
neurons communicate and synchronize with the periphery? Are peripheral clocks
in different tissues somewhat connected in an SCN-independent manner? Is there
any functional feedback from peripheral oscillators back to the SCN?

Solving
these points will be highly valuable for biomedical research. As the highly orchestrated
network of clocks is based on cascades of signaling pathways, studies by several
laboratories focused on understanding how clocks lead to the activation of transcriptional
programs that define the unique circadian features of a given tissue. The important
surprise came when transcriptional array profiles demonstrated that the clock
controls a remarkable fraction of the genome.

Circadian Genomics and Epigenomics

Since the original discovery of the period (per) gene in the fly by Ronald Konopka
and Seymour Benzer more than 40 years ago, the analysis of clock genes and their
relationships and functions has kept an increasing number of researchers busy.12At
the heart of the molecular network that constitutes the circadian clock are
factors involved in turning "on" or "off"’ transcription organized in feedback
loops. This organization ensures cycles of oscillatory gene expression and the
control of a remarkable fraction of the genome. Various studies have established that at least 10
percent of all expressed genes in any tissue are under circadian regulation.13Additional
levels of circadian regulation implicate parallel and intertwined regulatory
loops and the control by the cell of clock proteins stability. Moreover, scientists
anticipate that tissue-specific transcriptional
regulators contribute or intersect with the clock machinery.

The unexpectedly high proportion
of circadian transcripts suggests that the clock machinery may direct
widespread events of cyclic chromatin remodeling, which is the dynamic
modification of chromatin architecture to allow access of condensed genomic
DNA. Thisconsequently affects the cycles of
transcriptional activation and repression. Remarkably, a recent analysis
covering 14 types of mouse tissues identified approximately 10,000 known genes showing
circadian oscillations in at least one tissue.

These
findings underscore the presence of molecular interplays between the core clockwork,
which can be assumed to be common to all tissues, and cell-specific
transcriptional systems. Taking into consideration the recent view of the
mammalian circadian clock as a transcriptional network, through which the
oscillator acquires plasticity and robustness, it is reasonable to speculate
that the clock network contributes to physiological responses by intersecting
with cell-specific transcriptional pathways.13

Considering
the thousands of genes regulated in a circadian manner, researchers have questioned
how the complex organization of chromatin copes with the task of controlling
harmonic oscillations. A number of studies have revealed that several chromatin
dynamics contribute to circadian function, rendering specific genomic loci
either active (open) or silenced (closed) for transcription.13Specifically, we have found that the clock machinery is
itself essential for circadian control of chromatin dynamics.

This
finding provided a gateway to search for other components of the circadian
chromatin complexes.13 One of these is MLL1, an enzyme implicated in
some forms of cancer, that dictates the recruitment to chromatin of the clock
machinery thereby targeting circadian genes.14

As
soon as the first chromatin circadian regulators were identified, the search
for the counterbalancing enzymes was open. The discovery that the activity of SIRT1—a
longevity-associated enzyme belonging to a family of nicotinamide adenine
dinucleotide (NAD+) activated deacetylases—oscillates in a circadian
fashion established the first molecular and conceptual link between the
circadian clock and metabolism.15, 16 SIRT1 demonstrates an oscillation in activity, impinging back on the circadian
clock. The discovery of circadian-directed sirtuin activity spurred hypotheses
as to whether metabolites such as NAD+ themselves serve a predominant
role in the cellular link between metabolism and the circadian clock.15, 16

The Metabolic Clock

Intuitively,
circadian physiology implies that a considerable fraction of cellular
metabolism is cyclic. Also, the analysis of mice mutants for clock proteins has
revealed a number of metabolic defects. Indeed, metabolome analyses by mass
spectrometry have shown that about 50 percent of all metabolites oscillate in a
given tissue. Yet, the question that we have been addressing is as follows: What
is the molecular link between clock-driven control and the oscillation of a
given metabolite? In this respect, the example of NAD+ is
paradigmatic.

Indeed,
circadian oscillation of SIRT1 activity suggested that cellular NAD+
levels may oscillate. This is indeed the case, and the way this regulation is
achieved is conceptually revealing. The circadian clock controls the expression
of the gene encoding nicotinamide
phosphoribosyltransferase (NAMPT), a key rate-limiting enzyme in the
salvage pathway of NAD+ biosynthesis.17, 18 The clock
machinery is recruited to the NAMPT promoter in a time-dependent manner. The
oscillatory expression of NAMPT is abolished in mice mutated in clock function,
leading to drastically reduced and nonoscillatory levels of NAD+.
These results make a compelling case for the existence of an interlocking,
classical, transcriptional feedback loop that controls the circadian clock with
an enzymatic loop wherein SIRT1 regulates the levels of its own cofactor.17,
18

More
recently, we have questioned whether a nutritional challenge would modify the
genomic and metabolomic circadian profile. The nutritional implications of this
approach are multiple, especially in a modern society with an endless
availability of food. Mice that are fed a high-fat diet (HFD) experience a
drastic reprogramming of the circadian clock. Genes that normally would
oscillate stop doing so; in addition, many genes whose expression profile is
normally noncyclic start to oscillate.19 The HFD-induced
reprogramming pushes the liver to acquire a new circadian homeostasis that
implicates genes of the inflammasome and heat-shock response. In this sense,
the example of NAMPT and NAD+ is again very revealing. Under HFD, the
oscillation of both NAMPT and NAD+ is
abolished because the clock machinery cannot be recruited to chromatin. This illustrates
that different nutrition strategies directly “talk” to chromatin remodelers,
resulting in a reprogramming of genomic functions.19

The Next Phase

Mysteries in circadian biology remain. The
intrinsic, fundamental role played by the circadian clock in a large array of
biological functions illustrates that much more will be unraveled in the
upcoming years. Specifically, we predict that the clock will be found to play a
key role in the host-pathogen relationship, in the inflammatory response to
infection, and in the disturbances caused by tumoral growth. Based on our
current knowledge, it will be critical to decipher the role that specific
epigenetic regulators have in controlling the circadian epigenome.

Recent findings stress the role of two HDACs of
the sirtuin family, SIRT1 and SIRT6, in partitioning the circadian genome in
functional subdomains.20 This partitioning leads to a segregation of
cellular metabolism, again underscoring the intimate link with homeostasis.20
Finally, the role of circadian metabolism in neurons is likely to reveal yet-unexplored
regulation pathways that may help us decipher the relationship that the circadian
clock has with the sleep-wake cycle.

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About Cerebrum

Bill Glovin, editor Carolyn Asbury, Ph.D., consultant

Scientific Advisory Board Joseph T. Coyle, M.D., Harvard Medical School Kay Redfield Jamison, Ph.D., The Johns Hopkins University School of Medicine Pierre J. Magistretti, M.D., Ph.D., University of Lausanne Medical School and Hospital Robert Malenka, M.D., Ph.D., Stanford University School of Medicine Bruce S. McEwen, Ph.D., The Rockefeller University Donald Price, M.D., The Johns Hopkins University School of Medicine